scholarly journals Recombinant expression of lytic polysaccharide monooxygenase and its functional characterization

BioResources ◽  
2020 ◽  
Vol 15 (3) ◽  
pp. 7143-7158
Keyword(s):  
Cellulose Degradation ◽  
Synergistic Effects ◽  
Recombinant Expression ◽  
Critical Role ◽  
Functional Characterization ◽  
Maximal Activity ◽  
Enzymic Activity ◽  
Good Choice ◽  
Lytic Polysaccharide Monooxygenase ◽  
Flight Mass Spectrometry

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that can act on crystalline polysaccharides directly, which plays a critical role in cellulose degradation. In addition to reports on its structure and mechanism of action, it is important to study the auxiliary activity 9 (AA9) characteristics from different resources to support the mechanism research. The gene encoded ToLPMO9A was cloned from Trichoderma orientalis EU7-22 and first heterologously expressed in Pichia pastoris GS115. Both metal ions and reducing agent concentrations showed an important effect on ToLPMO9A. The ToLPMO9A exhibited maximal activity at 60 °C and a 6.0 pH. In addition, ToLPMO9A showed substrate specificity. The matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis showed that ToLPMO9A cleaved the glycosidic bonds at C1 and C4/C6 position via oxidation. Concerning the synergistic effects on enzymic activity, ToLPMO9A exhibited promotion with endo-glucanase or exo-glucanase, but inhibition with β-glucosidases. In conclusion, ToLPMO9A could be a good choice for enzyme cocktails and provide theoretical support for subsequent action mechanisms and broader applications.

scholarly journals Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 562
Author(s):  
Marlene Vuillemin ◽  
Artem S. Silchenko ◽  
Hang Thi Thuy Cao ◽  
Maxim S. Kokoulin ◽  
Vo Thi Dieu Trang ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Biological Activities ◽  
Functional Characterization ◽  
Maximal Activity ◽  
Brown Macroalgae ◽  
Pharmaceutical Uses ◽  
Enzymatic Depolymerization

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

scholarly journals Recombinant expression, biophysical and functional characterization of ClpS from Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Chenyun Guo ◽  
Yihang Xiao ◽  
Fangkai Bi ◽  
Weiliang Lin ◽  
Huilin Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mechanism ◽  
Recombinant Expression ◽  
Critical Role ◽  
Functional Characterization ◽  
High Yield ◽  
Titration Calorimetry ◽  
Screening Experiments ◽  
Intracellular Proteolysis

Abstract Intracellular proteolysis is attracting more and more attention for its unique and important character in Mycobacterium tuberculosis (Mt). The ClpS protein from Mt (MtClpS) plays a critical role in intracellular proteolysis by recognizing N-end rule substrates, which makes it become a potential target for antibacterial drugs. However, the molecular mechanism of MtClpS recognizing N-end rule substrates remains unclear. Preparation of highly concentrated and pure MtClpS protein is a prerequisite for further structural and functional studies. In the present work, we tried several fusion tags and various expression conditions to maximize the production of MtClpS in Escherichia coli. We established an efficient approach for preparing the MtClpS protein with a high yield of 24.7 mg/l and a high purity of 98%. After buffer screening, we obtained a stable MtClpS protein sample concentrated at 0.63 mM in the presence of glycerol, l-Arginine, and l-Glutamate. Moreover, circular dichroism characterization indicated that the secondary structure of MtClpS consists of 38% α-helix and 24% β-sheet. The 2D 1H-15N HSQC nuclear magnetic resonance spectrum showed a good dispersion of resonance peaks with uniform intensity, indicating that the purified MtClpS protein was well folded and conformationally homogeneous. Isothermal titration calorimetry experiments revealed significant interactions of MtClpS with N-end rule peptides beginning with Leu, Tyr, Trp, or Phe. Furthermore, residues D34, D35, and H66 were confirmed as key residues for MtClpS recognizing the N-end rule peptide. The successful expression and biophysical characterization of MtClpS enabled us to gain insight into the molecular mechanism of MtClpS recognizing N-end rule substrates. The obtained stable and pure recombinant MtClpS will enable future inhibitor screening experiments.

scholarly journals Deciphering the Mounting Complexity of the p53 Regulatory Network in Correlation to Long Non-Coding RNAs (lncRNAs) in Ovarian Cancer

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 527 ◽  
Author(s):  
Sonali Pal ◽  
Manoj Garg ◽  
Amit Kumar Pandey
Keyword(s):  
Ovarian Cancer ◽  
Molecular Mechanisms ◽  
Human Cancer ◽  
Critical Role ◽  
Functional Characterization ◽  
Great Promise ◽  
Altered Expression ◽  
Disease Biology ◽  
Non Coding Rnas ◽  
Post Transcriptional Regulation

Amongst the various gynecological malignancies affecting female health globally, ovarian cancer is one of the predominant and lethal among all. The identification and functional characterization of long non-coding RNAs (lncRNAs) are made possible with the advent of RNA-seq and the advancement of computational logarithm in understanding human disease biology. LncRNAs can interact with deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins and their combinations. Moreover, lncRNAs regulate orchestra of diverse functions including chromatin organization and transcriptional and post-transcriptional regulation. LncRNAs have conferred their critical role in key biological processes in human cancer including tumor initiation, proliferation, cell cycle, apoptosis, necroptosis, autophagy, and metastasis. The interwoven function of tumor-suppressor protein p53-linked lncRNAs in the ovarian cancer paradigm is of paramount importance. Several lncRNAs operate as p53 regulators or effectors and modulates a diverse array of functions either by participating in various signaling cascades or via interaction with different proteins. This review highlights the recent progress made in the identification of p53 associated lncRNAs while elucidating their molecular mechanisms behind the altered expression in ovarian cancer tumorigenesis. Moreover, the development of novel clinical and therapeutic strategies for targeting lncRNAs in human cancers harbors great promise.

scholarly journals Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
María-Eugenia DeCastro ◽  
Michael P. Doane ◽  
Elizabeth Ann Dinsdale ◽  
Esther Rodríguez-Belmonte ◽  
María-Isabel González-Siso
Keyword(s):  
Microbial Community ◽  
Hot Spring ◽  
Functional Characterization ◽  
Tca Cycle ◽  
Maximal Activity ◽  
Sulfur Cycle ◽  
E Coli ◽  
Complex Microbial Community ◽  
Relationship Of ◽  
The Relationship

AbstractIn the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin–Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-β-d-galactopyranoside (ONPG) and p-Nitrophenyl-β-d-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.

Molecular and Functional Characterization of One Odorant-Binding Protein Gene OBP3 in Bemisia tabaci (Hemiptera: Aleyrodidae)

2019 ◽  
Author(s):  
Ran Wang ◽  
Yuan Hu ◽  
Peiling Wei ◽  
Cheng Qu ◽  
Chen Luo
Keyword(s):  
Host Plant ◽  
Bemisia Tabaci ◽  
Binding Protein ◽  
Insect Pest ◽  
Critical Role ◽  
Functional Characterization ◽  
Odorant Binding Protein ◽  
Binding Characteristics ◽  
And Function ◽  
Odorant Binding

Abstract Odorant binding proteins (OBPs) of insects play a critical role in chemical perceptions and choice of insect host plant. Bemisia tabaci is a notorious insect pest which can damage more than 600 plant species. In order to explore functions of OBPs in B. tabaci, here we investigated binding characteristics and function of odorant-binding protein 3 in B. tabaci (BtabOBP3). The results indicated that BtabOBP3 shows highly similar sequence with OBPs of other insects, including the typical signature motif of six cysteines. The recombinant BtabOBP3 protein was obtained, and the evaluation of binding affinities to tested volatiles of host plant was conducted, then the results indicated that β-ionone had significantly higher binding to BtabOBP3 among other tested plant volatiles. Furthermore, silencing of BtabOBP3 significantly altered choice behavior of B. tabaci to β-ionone. In conclusion, it has been demonstrated that BtabOBP3 exerts function as one carrier of β-ionone and the results could be contributed to reveal the mechanisms of choosing host plant in B. tabaci.

scholarly journals Proteomics of Culture Filtrate of Prevalent Mycobacterium tuberculosis Strains: 2D-PAGE Map and MALDI-TOF/MS Analysis

2017 ◽  
Vol 22 (9) ◽  
pp. 1142-1149 ◽  
Author(s):  
Gavish Kumar ◽  
Hari Shankar ◽  
Divakar Sharma ◽  
Prashant Sharma ◽  
Deepa Bisht ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Culture Filtrate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Critical Role ◽  
Laboratory Strain ◽  
Proteomic Profiling ◽  
East African ◽  
Mycobacterium Tuberculosis H37rv ◽  
Flight Mass Spectrometry ◽  
Central Asian

Although diverse efforts have been done to identify biomarkers for control of tuberculosis using laboratory strain Mycobacterium tuberculosis H37Rv, the disease still poses a threat to mankind. There are many emerging M. tuberculosis strains, and proteomic profiling of these strains might be important to find out potential targets for diagnosis and/or prevention of tuberculosis. We evaluated the comparative proteomic profiling of culture filtrate (CF) proteins from prevalent M. tuberculosis strains (Central Asian or Delhi type; CAS1_Del, East African-Indian; EAI-3 and Beijing family) by 2D polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. As a result, we could identify 12 CF proteins (Rv0066c, Rv1310, Rv3375, Rv1415, Rv0567, Rv1886c, Rv3803c, Rv3804c, Rv2031c, Rv1038c, Rv2809, and Rv1911c), which were consistently increased in all prevalent M. tuberculosis strains, and interestingly, two CF proteins (Rv2809, Rv1911c) were identified with unknown functions. Consistent increased intensity of these proteins suggests their critical role for survival of prevalent M. tuberculosis isolates, and some of these proteins may also have potential as diagnostic and vaccine candidates for tuberculosis, which needs to be further explored by immunological analysis.

Dynamic regulation of the voltage-gated Kv2.1 potassium channel by multisite phosphorylation

2007 ◽  
Vol 35 (5) ◽  
pp. 1064-1068 ◽  
Author(s):  
D.P. Mohapatra ◽  
K.-S. Park ◽  
J.S. Trimmer
Keyword(s):  
Neuronal Excitability ◽  
Critical Role ◽  
Functional Characterization ◽  
K Channel ◽  
Delayed Rectifier ◽  
Dynamic Regulation ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Specific Regulation

Voltage-gated K+ channels are key regulators of neuronal excitability. The Kv2.1 voltage-gated K+ channel is the major delayed rectifier K+ channel expressed in most central neurons, where it exists as a highly phosphorylated protein. Kv2.1 plays a critical role in homoeostatic regulation of intrinsic neuronal excitability through its activity- and calcineurin-dependent dephosphorylation. Here, we review studies leading to the identification and functional characterization of in vivo Kv2.1 phosphorylation sites, a subset of which contribute to graded modulation of voltage-dependent gating. These findings show that distinct developmental-, cell- and state-specific regulation of phosphorylation at specific sites confers a diversity of functions on Kv2.1 that is critical to its role as a regulator of intrinsic neuronal excitability.

scholarly journals Avian Pattern Recognition Receptor Sensing and Signaling

2020 ◽  
Vol 7 (1) ◽  
pp. 14 ◽  
Author(s):  
Sabari Nath Neerukonda ◽  
Upendra Katneni
Keyword(s):  
Pattern Recognition ◽  
Rational Design ◽  
Animal Species ◽  
Critical Role ◽  
Functional Characterization ◽  
Structural Features ◽  
Present Review ◽  
Vaccine Adjuvants ◽  
Microbial Defense ◽  
Recognition Receptor

Pattern recognition receptors (PRRs) are a class of immune sensors that play a critical role in detecting and responding to several conserved patterns of microorganisms. As such, they play a major role in the maintenance of immune homeostasis and anti-microbial defense. Fundamental knowledge pertaining to the discovery of PRR functions and their ligands continue to advance the understanding of immune system and disease resistance, which led to the rational design and/or application of various PRR ligands as vaccine adjuvants. In addition, the conserved nature of many PRRs throughout the animal kingdom has enabled the utilization of the comparative genomics approach in PRR identification and the study of evolution, structural features, and functions in many animal species including avian. In the present review, we focused on PRR sensing and signaling functions in the avian species, domestic chicken, mallard, and domestic goose. In addition to summarizing recent advances in the understanding of avian PRR functions, the present review utilized a comparative biology approach to identify additional PRRs, whose functions have been well studied in mammalians but await functional characterization in avian.

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